Capillary electrophoresis is an analytical tool wherein analyte ions in an electrolyte solution are separated using an electric field. A source of voltage is coupled between the input (or source) and output (or destination) ends of a narrow bore capillary containing the electrolyte solution. The voltage source establishes a voltage gradient axially along the capillary that causes axial migration of ions within the capillary.
Typically the capillary is made from silica, a material that forms fixed negative charges on the inner capillary wall in the presence of an electrolyte solution. Even before the voltage gradient is applied, cations in the electrolyte solution will be attracted to these fixed negative charges, forming a so-called double layer at the capillary wall. Because they are negatively charged, electrolyte anions will be repelled from the capillary wall.
In a commonly used mode of operation, the voltage gradient is applied such that the source end of the capillary is positive relative to the destination end. Electrolyte cations are attracted both to the negatively charged capillary wall and to the capillary end coupled to the negative end of the voltage gradient. Conversely, electrolyte anions are repelled from the capillary wall and are attracted to the capillary end coupled to the positive end of the voltage gradient.
The voltage gradient will create a net movement of the cations loosely associated with the fixed negative charges at the electrolyte/silica interface, which movement drags the bulk of the electrolyte solution toward the negatively charged capillary destination end. This net bulk flow towards one capillary end is termed electroendosmotic or electroosmotic flow and has a characteristic plug-like velocity profile radially across the capillary. By plug-like, it is meant that a radial cross-section of the flow will exhibit uniform velocity everywhere.
The bulk electrolyte solution movement occurs at a rate proportional to electroosmotic mobility, .mu..sub.eo. Generally, mobility is the per unit time rate of movement toward a charge, normalized for the magnitude of the imposed electrical field that induces the movement. Electroosmotic flow is further characterized by the absence of an axial pressure differential across the capillary through which the flow is occurring.
As cations subjected to the voltage gradient cause the electrolyte solution to move axially along the capillary length due to electroosmotic flow, variously charged analyte ions that might be present within the electrolyte solution are subjected to electrophoresis. Electrophoresis is the movement of analyte ions in an axial direction, toward an electrode of opposite charge or away from an electrode of like charge at the capillary ends.
The rate of electrophoretic movement is influenced by the axial voltage gradient (i.e. the electrical field strength) imposed across the length of the capillary through the electrolyte solution, and by the analyte electrophoretic mobility, .mu..sub.a. Analyte electrophoretic mobility is the per unit time rate of axial movement of an ion per unit electrical field strength toward or away from appropriately charged electrodes. The rate of electrophoretic movement is defined in the absence of any electroosmotic flow.
Analyte ions within the electrolyte solution migrate differentially based upon their electrophoretic mobilities. If, for example, the rate of the electroosmotic flow exceeds the rate of electrophoretic movement of anions, all of the analyte ions are moved axially in the direction of the electroosmotic flow, but at different rates. In a capillary with a negatively-charged inner wall, anions tend to move in a direction opposite to the electroosmotic flow, but may be swept along in the direction of the prevailing electroosmotic flow. By contrast, cations move in the same direction as the electroosmotic flow and thus are swept along more rapidly than anions. Neutral species are carried along solely by the electroosmotic flow and migrate together as a band situated after the faster-moving cation bands but before slower-moving anion bands.
In another mode of operation, it is also possible to reverse the negative charge on the capillary wall by suitably modifying the silica wall such that fixed positive charges are present. In this mode, anions in the electrolyte are attracted to the charged wall, again forming an electrical double layer. Electrolyte anions then move toward the positive end of the electric field, which is coupled to the destination end of the capillary. In this mode, the anions migrate in the same direction as the bulk electroosmotic flow.
In either mode, for ease of analysis it is desirable that an analyte traverse the capillary length at a constant reproducible time, the so-called migration time. A constant migration time for a given set of conditions (e.g., electrolyte solution composition, pH, concentration, temperature, and electric field strength) simplifies analysis since analyte peak areas need not be corrected for migration time differences. Further, computerized peak identification of analytes is dependent upon relatively little or no change in migration times in sequential separations of standards and unknown samples.
Migration time depends upon the velocity of the analyte relative to the inner capillary wall, e.g., the net velocity of the analyte. Within the capillary, net analyte velocity represents the product of the electric field strength E multiplied by the sum of the analyte mobility .mu..sub.a for each analyte species and the electroosmotic mobility .mu..sub.eo.
While analyte mobility .mu..sub.a is invariant for a given set of conditions, electroosmotic mobility .mu..sub.eo is dependent upon the potential appearing at the surface of the inner capillary wall, the so-called zeta potential. The zeta potential can vary with time as slow chemical changes take place at the capillary surface. For example, reequilibration of the capillary after cleaning, adsorption of contaminant molecules, and slow desorption of contaminant molecules from the capillary wall can affect the zeta potential, thus affecting electroosmotic mobility, .mu..sub.eo.
If the electroosmotic mobility or flow can be measured and controlled, a constant analyte migration time and simplified analysis can result. Furthermore, control of electroosmotic flow can be used to optimize separation of mixtures of analyte molecules by permitting adjustment of migration times. For example, slowing flow during the separation of high-mobility analytes will permit a longer time period to achieve better separation of analyte molecules with similar electrophoretic mobility values.
Electroosmotic flow measurement and control requires measurement of the instantaneous electroosmotic mobility or flow, whereupon the electroosmotic mobility may be adjusted to restore its original value. If the instantaneous electroosmotic flow ate is known, it may be suitably adjusted to provide the desired constant analyte migration time. As used herein, the terms electroosmotic flow and electroosmotic mobility will be used interchangeably. This interchangeability of terms is justified since electroosmotic flow=(electroosmotic mobility).multidot.(electrical field strength).multidot.(cross-sectional capillary area).
Measurement of the electroosmotic flow may be used to gauge the state of the separation capillary, as the flow directly reflects the extent of contamination or change of the charge layer on the capillary wall. Electroosmotic flow also can serve as an internal reference for expressing a migration parameter for analyte species, analogous to the way the dead time or dead volume is used to derive capacity factor k' in chromatographic separations.
Various techniques are practiced in the prior art to determine electroosmotic flow. It is known to use an electrically neutral flow marker to measure electroosmotic flow, see for example, T. S. Stevens and H. J. Cortes, Analytical Chemistry (1983) 55 1365-1370; K. D. Lukacs and J. W. Jorgenson, J. High Resolut. Chromatogr. Chromatogr. Commun (1985) 8 407; J. E. Wiktorowicz, U.S. Pat. Nos. 5,181,999 and 5,015,350. Hanai et al. (J. High Resolut. Chromatogr. Chromatogr. Commun. (1991) 14 483) disclosed the use of a number of neutral flow markers for the determination of electroosmotic flow rates in capillary electrophoresis. In addition, Lee et al. (J. Chromatogr. (1991) 559 122-140) described the use of a UV marker that was injected into the capillary to monitor changes in electroosmotic flow induced by application of an external electrical field.
However, it is difficult to find a neutral marker species readily soluble in aqueous solutions that has the requisite spectral characteristics to render it detectable by conventional capillary electrophoresis ("CE") detection technology. The marker must be truly neutral under all pH conditions, and must not adsorb or partition onto the capillary wall. The UV marker method permits only one measurement of the electroosmotic flow during each electrophoretic separation, and is not a continuous, realtime measurement method.
While the use of multiple series-connected conductivity detectors to measure analyte mobility .mu..sub.a is known, such technique cannot measure electroosmotic mobility .mu..sub.eo. See J. L. Beckers, Th. P. E. M. Vergeggen, F. M. Everaerts, J. Chromatogr. (1988) 452 591-600.
Another prior art approach measures electroosmotic flow by measuring changes in electrical current flowing through the capillary as electroosmotic flow displaces the electrolyte solution therein with an electrolyte solution of slightly different composition. See, for example, Huang, Gordon, and Zare, Analytical Chemistry (1988) 60 1837-1838. In Analytical Chemistry (1991) 63 1519-1523, Lee, et al. describe the use of the above-described current-monitoring method to monitor changes in electroosmotic flow induced in a separation capillary by application of an external electric field.
However, this "off-line" current-monitoring technique does not permit real-time measurements, e.g., measurements made contemporaneously during actual separation of analyte ions. Further, this prior art measurement technique will by necessity change the chemical composition of the electrolyte solution in the capillary. As such, this technique interferes with the accuracy of the measurement of the magnitude of electroosmotic flow, which is a function of both the ionic strength and the chemical composition of the electrolyte solution.
Measuring the time required for a change in electrolyte solution concentration to reach the detector has also been used to determine electroosmotic mobility .mu..sub.eo ; see, for example U.S. Pat. No. 5,009,760 to Zare. However, as noted, such measurements are not accurate in that the electrolyte solution is changed during the measurement process. Further, such measurements do not provide the instantaneous electroosmotic flow rate measurement required to allow rapid flow rate adjustment.
Yet another prior art technique requires the periodic weighing or volumetric determination of the quantity of effluent electrolyte solution from the capillary. The electroosmotic flow rate is estimated from the mass flow rate of the electrolyte. This technique is described by Altria and Simpson, Analytical Proceedings (1986) 23 453 and B. J. Wanders, A. A. A. M. Van de Goor, and F. M. Everaerts, J. Chromatogr. (1989) 470 89-93. Van de Goor et al. subsequently teach the use of on-line weighing of the effluent from the separation capillary as a method of deriving knowledge of the electroosmotic flow rate through the capillary (A. A. A. M. Van de Goor, B. J. Wanders, and F. M. Everaerts, J. Chromatogr. (1989) 95-104). However, measurements of instantaneous electroosmotic flow were not possible as readings could be taken only every 5 minutes.
Lee and Hong (J. Membrane Sci. (1988) 39 79-88) have described a device for measurement of electroosmotic flow through microporous membranes. The Lee-Hong device is based on measurement of the volume of liquid pumped out of an overflow channel from a chamber into which fluid is being pumped via electroosmotic flow. The volume of fluid pumped out of the overflow channel as a function of time is a measure of the magnitude of rate of electroosmotic flow of fluid into the chamber. Generally, however, such measurements cannot be conducted in real-time during an actual separation of the analyte ions with sufficient frequency as to permit control of the electroosmotic flow during the separation.
Van de Goor et al. also teach the use of off-line measurements of streaming potential to observe changes in the capillary with time. Similarly, Wang and Hartwick (J. Chromatogr. 594 1992 325-334) and Reijenga et al. (J. Chromatogr 260 (1983) 241-254) teach the use of streaming potential measurement for characterization of the capillary wall in contact with various electrolyte solutions. No real-time, on-line measurement of electroosmotic flow through a separation capillary was discussed by either reference.
One real-time, on-line prior art approach to monitoring electroosmotic flow introduces a hydrostatically-driven liquid stream containing an ultraviolet-absorbing species into the electroosmotically-moved electrolyte solution from the separation capillary. Under constant electroosmotic flow and hydrostatic flow conditions, a combined effluent stream of constant ultraviolet absorptivity results. Electroosmotic flow rate variations produce changes in the concentration of the ultraviolet-absorbing tracer species in the combined effluent stream. Real-time electroosmotic flow data can be obtained, permitting use of feedback to control electroosmotic mobility by changing the magnitude of the axial voltage gradient along the separation capillary such that migration times are made more constant; see Wanders, Van de Goor, and Everaerts, J. Chromatogr. (1989) 470 89-93.
Understandably, providing a suitable mechanism for combining the two streams and sensing ultraviolet concentration complicates the measurement process. In addition, changes in the hydrostatically-driven flow of UV absorbing tracer as well as a change in the electroosmotic flow rate can result in a change in the spectral absorbance of the combined stream of effluent. Finally, though this approach can vary electroosmotic flow by changing the axial voltage gradient along the capillary, the approach is of moderate value in practice. Variation of the axial voltage gradient produces analyte electrophoretic velocity changes concomitant with changes in the electroosmotic velocity.
Finally, in a scientific field unrelated to capillary electrophoresis, Miyamoto, et al., J. Membrane Sci (1989) 41 377-391, describe measurement of electroosmotic flow through sections of frog skin and gastric mucosa membranes. Miyamoto measured electroosmotic flow using a photodiode positioned along the overflow channel from a chamber into which fluid was pumped electroosmotically. Use of a pressure transducer to measure the flow indirectly was also described, wherein fluid pressure drop along the channel due to fluid flow into the chamber was measured.
The majority of the above-mentioned measuring methods are not readily amenable to on-line measurement of electroosmotic flow through separation capillaries. These prior art methods either do not provide a continuous, instantaneous measurement (e.g., use of a neutral flow marker) or they perturb the electroosmotic flow in the measurement process (e.g., measurement of the rate of change of a different buffer electroosmotically-pumped into the capillary).
What is needed is an on-line, real-time method and apparatus for monitoring and measuring electroosmotic flow that do not alter the electrolyte solution under measurement, and that do not impose substantial hardware overhead. Preferably such method and apparatus should produce a signal proportional to the magnitude of the electroosmotic flow, which signal may be used (manually or in a feedback loop) to adjust conditions such that electroosmotic flow can be made constant or changed in a predictable manner.
The present invention discloses such methods and apparatus.